Generation of induced neurons via direct conversion in vivo and in vitro
(2014) In Lund University Faculty of Medicine Doctoral Dissertation Series 2014:20.- Abstract
- Cellular reprogramming is when one cell is changed into another. This involves structural modifications on the DNA of a cell
resulting in a transcriptional change. This occurs naturally during development when early pluripotent cells gradually differentiate into more
specialized cells that finally result in a complete organism. This is a finely orchestrated event that includes both extrinsic and intrinsic signaling.
Cellular reprogramming can be induced artificially by exposing a somatic cell to a foreign microenvironment or by the forced expression
of various transcription factors. Recent studies have shown the possibility to revert a somatic cell back into a pluripotent stem cell, termed
... (More) - Cellular reprogramming is when one cell is changed into another. This involves structural modifications on the DNA of a cell
resulting in a transcriptional change. This occurs naturally during development when early pluripotent cells gradually differentiate into more
specialized cells that finally result in a complete organism. This is a finely orchestrated event that includes both extrinsic and intrinsic signaling.
Cellular reprogramming can be induced artificially by exposing a somatic cell to a foreign microenvironment or by the forced expression
of various transcription factors. Recent studies have shown the possibility to revert a somatic cell back into a pluripotent stem cell, termed
induced pluripotent stem cell (iPS) or directly to a different somatic cell using this strategy.
In this thesis I focus on the direct reprogramming where one terminally differentiated cell is directly converted into another without passing
a pluripotent state. Using lentiviral vectors we could convert embryonic and postnatal human fibroblasts into functional neurons (iN) by the
forced expression of Ascl1, Brn2 and Myt1L (ABM). By including the additional factors, Foxa2 and Lmx1a, subtype specific neurons could be
obtained that release dopamine, express specific markers and exhibit electrophysiological properties characteristic of dopaminergic neurons.
Further we show the possibility to transplant fibroblasts and astrocytes into brains of adult rats and then convert them into neurons in vivo.
These cells expressed pan- neuronal markers and converted at similar rates as reported in vitro. Using Cre inducible lentiviral vectors, coding
for ABM and inject these into the brains of transgenic mice expressing Cre under the GFAP promoter, we could specifically target astrocytes and
convert these into neurons in vivo. Using the same strategy we cloned the three factors, Ascl1, Lmx1a and Nurr1 (ALN) together with GFP, into
Cre inducible recombinant adeno associated viral vectors (rAAV) with the aim to convert NG2 glia into dopaminergic neurons. rAAV vectors
are interesting tools for clinical applications because of their low pathogenicity and their ability to infect both dividing and non-dividing cells.
By including a synapsin promoter for the GFP reporter we could specifically visualize converted cells that expressed the pan neuronal markers
NeuN and MAP2 but failed to induce a dopaminergic phenotype. More studies aim to study these cells after a longer maturation time and their
functional properties in terms of electrophysiology and synaptic formation.
Cellular reprogramming of somatic cells is an interesting option to previously studied sources in cell replacement therapies that often are
associated with logistical and ethical concerns. They are readily available cells that can be obtained from the skin of a patient and direct conversion
offers further advantages over iPS cells as they are non-proliferating cells eliminating the risk of forming tumors when transplanted.
Further, in vivo reprogramming offers an alternative to traditional cell therapy by creating new neurons in the brain removing the need of an
exogenous cell source. The brain is of particular interest for cell replacement therapies as its capacity to repair itself after injuries like stroke
is limited and treatments for neurological disorders like Parkinson’s disease (PD) progressively decline in effectiveness and are associated
with severe side effects.
In summary, this thesis shows the possibility to directly convert human, adult fibroblasts into functional dopaminergic neurons by the forced
expression of transcription factors important in neural development. We further show the possibility to transplant fibroblasts and astrocytes
into the brains of rats and convert them into neurons in situ. We also show the possibility to convert two types of glia cells, astrocytes and NG2
glia residing in the brain into neurons by using transgenic mice and Cre inducible vectors. This could also be done by using a rAAV vector commonly
used in clinical trials. Future studies should focus on factors involved in the specificity of the required cell and how well the cell that is
formed correspond genetically, functionally and viably to its endogenous counterpart. (Less) - Abstract (Swedish)
- Popular Abstract in Swedish
I min avhandling har jag tittat på möjligheten att göra om mänskliga hudceller
direkt till dopaminerga nervceller. Dessa uppvisar flera egenskaper tillskrivna de
dopaminerga nervceller vi hittar i hjärnan. Jag tittar även på möjligheten att transplantera
dessa som hudceller i hjärnan på råttor och sen göra om dom till nervceller
när de är på plats. Dessa celler bildar nervceller i samma utsträckning som när de
görs om i cellkultur. Sist riktar vi in oss på två olika celltyper i hjärnan som inte är
nervceller och gör om dessa direkt till nervceller på plats i hjärnan. Detta kan tillföra
ännu en dimension till cellterapi då nya celler... (More) - Popular Abstract in Swedish
I min avhandling har jag tittat på möjligheten att göra om mänskliga hudceller
direkt till dopaminerga nervceller. Dessa uppvisar flera egenskaper tillskrivna de
dopaminerga nervceller vi hittar i hjärnan. Jag tittar även på möjligheten att transplantera
dessa som hudceller i hjärnan på råttor och sen göra om dom till nervceller
när de är på plats. Dessa celler bildar nervceller i samma utsträckning som när de
görs om i cellkultur. Sist riktar vi in oss på två olika celltyper i hjärnan som inte är
nervceller och gör om dessa direkt till nervceller på plats i hjärnan. Detta kan tillföra
ännu en dimension till cellterapi då nya celler kan skapas på plats i hjärnan och
externa källor för celler inte behövs.
Våra kroppsceller innehåller identisk genetisk information i form av DNA packat
i 23 st kromosompar. Cellerna skiljer sig åt i vilken genetisk information som faktiskt
uttrycks genom olika kemiska modifikationer på kromosomerna. Dessa modifieringar
styrs av olika signaler, bla av särskilda proteiner kallade transkriptionsfaktorer.
Genom att tvinga en cell till att uttrycka vissa transkriptionsfaktorer kan
man ändra de kemiska modifikationerna och på så vis göra om cellen till en annan.
Det kallas cellulär omprogrammering. På så vis har man gjort om vanliga hudceller
till en ur-stamcell med förmågan att bilda alla de olika celler som utgör en individ.
Man har även lyckats att göra om en hudcell direkt till en nervcell.
Varför är då detta intressant? Förutom att det visar på våra cellers enorma kapacitet
att inta olika roller beroende på vilka gener som uttrycks så finns det ett
medicinskt intresse. I det som kallas cell-terapi ämnar man ersätta sjuka eller döda
celler med nya genom transplantation. Detta är speciellt intressant för hjärnan
eftersom dess förmåga att laga sig själv efter skador såsom stroke (Sv. slaganfall)
är begränsad. Många sjukdomar som drabbar det centrala nervsystemet är också
intressanta kandidater för cellterapi. I Parkinsons sjukdom (PD) dör en specifik
sorts nervceller kallade dopaminerga nervceller vilka frisätter signalsubstansen
dopamin i hjärnan. Detta leder till svårigheter att utföra medvetna handlingar och
muskulär stelhet. Det finns idag ingen beständig behandling för patienter med PD
och de mediciner som ges tappar i effekt med tiden och besvärliga biverkningar
uppstår. Försök med patienter med PD till vilka man transplanterat fetal vävnad
innehållande de celler som så småningom bildar de dopminerga nervcellerna har
visat att det går att lindra symptomen förknippade med PD. Tillgången på fetal
vävnad är dock begränsad och det råder en del etiska kontroverser kring hanteringen
av den. En annan typ av celler man undersöker är embryonala stam celler.
Dessa kan isoleras från ett tidigt stadium av det befruktade ägget och har förmågan
att bilda alla celler i kroppen. Genom att styra dom till att bli dopaminerga nervceller
skulle de kunna användas i transplantationer till patienter med PD. Det finns
dock risker assoccierade med tumörbildningar i användandet av dessa celler då de
aktivt delar sig samt att vissa etiska kontroverser återstår.
20
Hudceller som direkt görs om till nervceller kringgår flera av dessa problem.
De är enkla att isolera från individen och eftersom de inte passerar ett stamcells
stadie utgör de ingen risk i bildandet av tumörer. Eftersom cellerna kan tas direkt
från patienten själv begränsar man även risken för bortstötning då cellerna är immunomatchade
till individen. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/4276198
- author
- Torper, Olof LU
- supervisor
-
- Malin Parmar LU
- Johan Jakobsson LU
- Anders Björklund LU
- opponent
-
- Professor Hermansson, Ola, Department of Neuroscience, Karolinska institutet, Retzius väg 8 S-171 77 Stockholm, Sweden
- organization
- publishing date
- 2014
- type
- Thesis
- publication status
- published
- subject
- keywords
- Cellular reprogramming, cell replacement therapies, induced dopaminergic neurons, in vivo reprogramming, Lentiviral vectors, AAV vectors
- in
- Lund University Faculty of Medicine Doctoral Dissertation Series
- volume
- 2014:20
- pages
- 125 pages
- publisher
- Neurobiology
- defense location
- Segerfalksalen, Wallenberg Neuroscience Center, Lund, Sweden
- defense date
- 2014-02-28 13:00:00
- ISSN
- 1652-8220
- ISBN
- 978-91-87651-44-1
- language
- English
- LU publication?
- yes
- id
- b3b72c69-3e85-4faa-b9d7-803e085aa7c0 (old id 4276198)
- date added to LUP
- 2016-04-01 13:55:12
- date last changed
- 2023-04-18 20:25:52
@phdthesis{b3b72c69-3e85-4faa-b9d7-803e085aa7c0,
abstract = {{Cellular reprogramming is when one cell is changed into another. This involves structural modifications on the DNA of a cell<br/><br>
resulting in a transcriptional change. This occurs naturally during development when early pluripotent cells gradually differentiate into more<br/><br>
specialized cells that finally result in a complete organism. This is a finely orchestrated event that includes both extrinsic and intrinsic signaling.<br/><br>
Cellular reprogramming can be induced artificially by exposing a somatic cell to a foreign microenvironment or by the forced expression<br/><br>
of various transcription factors. Recent studies have shown the possibility to revert a somatic cell back into a pluripotent stem cell, termed<br/><br>
induced pluripotent stem cell (iPS) or directly to a different somatic cell using this strategy.<br/><br>
In this thesis I focus on the direct reprogramming where one terminally differentiated cell is directly converted into another without passing<br/><br>
a pluripotent state. Using lentiviral vectors we could convert embryonic and postnatal human fibroblasts into functional neurons (iN) by the<br/><br>
forced expression of Ascl1, Brn2 and Myt1L (ABM). By including the additional factors, Foxa2 and Lmx1a, subtype specific neurons could be<br/><br>
obtained that release dopamine, express specific markers and exhibit electrophysiological properties characteristic of dopaminergic neurons.<br/><br>
Further we show the possibility to transplant fibroblasts and astrocytes into brains of adult rats and then convert them into neurons in vivo.<br/><br>
These cells expressed pan- neuronal markers and converted at similar rates as reported in vitro. Using Cre inducible lentiviral vectors, coding<br/><br>
for ABM and inject these into the brains of transgenic mice expressing Cre under the GFAP promoter, we could specifically target astrocytes and<br/><br>
convert these into neurons in vivo. Using the same strategy we cloned the three factors, Ascl1, Lmx1a and Nurr1 (ALN) together with GFP, into<br/><br>
Cre inducible recombinant adeno associated viral vectors (rAAV) with the aim to convert NG2 glia into dopaminergic neurons. rAAV vectors<br/><br>
are interesting tools for clinical applications because of their low pathogenicity and their ability to infect both dividing and non-dividing cells.<br/><br>
By including a synapsin promoter for the GFP reporter we could specifically visualize converted cells that expressed the pan neuronal markers<br/><br>
NeuN and MAP2 but failed to induce a dopaminergic phenotype. More studies aim to study these cells after a longer maturation time and their<br/><br>
functional properties in terms of electrophysiology and synaptic formation.<br/><br>
Cellular reprogramming of somatic cells is an interesting option to previously studied sources in cell replacement therapies that often are<br/><br>
associated with logistical and ethical concerns. They are readily available cells that can be obtained from the skin of a patient and direct conversion<br/><br>
offers further advantages over iPS cells as they are non-proliferating cells eliminating the risk of forming tumors when transplanted.<br/><br>
Further, in vivo reprogramming offers an alternative to traditional cell therapy by creating new neurons in the brain removing the need of an<br/><br>
exogenous cell source. The brain is of particular interest for cell replacement therapies as its capacity to repair itself after injuries like stroke<br/><br>
is limited and treatments for neurological disorders like Parkinson’s disease (PD) progressively decline in effectiveness and are associated<br/><br>
with severe side effects.<br/><br>
In summary, this thesis shows the possibility to directly convert human, adult fibroblasts into functional dopaminergic neurons by the forced<br/><br>
expression of transcription factors important in neural development. We further show the possibility to transplant fibroblasts and astrocytes<br/><br>
into the brains of rats and convert them into neurons in situ. We also show the possibility to convert two types of glia cells, astrocytes and NG2<br/><br>
glia residing in the brain into neurons by using transgenic mice and Cre inducible vectors. This could also be done by using a rAAV vector commonly<br/><br>
used in clinical trials. Future studies should focus on factors involved in the specificity of the required cell and how well the cell that is<br/><br>
formed correspond genetically, functionally and viably to its endogenous counterpart.}},
author = {{Torper, Olof}},
isbn = {{978-91-87651-44-1}},
issn = {{1652-8220}},
keywords = {{Cellular reprogramming; cell replacement therapies; induced dopaminergic neurons; in vivo reprogramming; Lentiviral vectors; AAV vectors}},
language = {{eng}},
publisher = {{Neurobiology}},
school = {{Lund University}},
series = {{Lund University Faculty of Medicine Doctoral Dissertation Series}},
title = {{Generation of induced neurons via direct conversion in vivo and in vitro}},
volume = {{2014:20}},
year = {{2014}},
}